SITE DIRECTED MUTAGENESIS

SITE DIRECTED MUTAGENESIS

GUIDED BY:- MR. DEVENDRA J. VAISHNAV

PRESENTED BY:- MR. YOGESH GHODASARAWhat we are studied hereDefinationWhy modify gene ?PrincipleMethod for MutagenesisApplicationAlternative method

DEFINATION MUTATION A Change in the genetic material of an organism.

DIRECTED MUTATION/SPECIFIC MUTATION Mutation at a specific predetermined location.4Why Modify the Gene? Why not Modify the Protein?If the gene is modified by site directed mutagenesis then each time the host organism will produce the modified protein.However if the protein is modified each time the protein is produced it has to be modified. Further more chemical modification of protein is:Harsh NonspecificHas to be repeatedly doneWhy Modify the Gene? Why not Modify the Protein?Protein engineering involves the use of genetic manipulations to alter the coding sequence of a (cloned) gene and thus modify the properties of the protein encoded by that gene.This mutant gene maybe expressed in a suitable system to produce unlimited quantities of the modified protein.Therefore site directed mutagenesis and protein engineering are used to change ( modify) the properties of a protein.

6What Properties of a Protein Would You Want to Change?We may be able to alter:Michaelis constant KmVmaxThermal stabilitypH stabilityCofactor requirementSpecificity

7Km/VmaxWhat is the Km of an enzyme ?Michaelis constant or Km is the tightness of the substrate binding to the enzyme.(increases the specificity of the reaction and reduce side reactions).

The Vmax is the maximal rate of conversion of the substrate to the products.(an increase in Vmax increase the amount of product produced).

An increase in pH or thermal stability may allow the protein to be used under conditions where it would normally be denatured.8Cofactor Requirement and Increase SpecificityThe abolishment of the need for a cofactor may be beneficial where under certain industrial conditions a cofactor has to be constantly provided.Increase specificity of the enzyme decreases undesirable side reactions.Principle

Denature methylated template and anneal divergent mutagenic primers.PCR amplify the entire plasmid with a DNA pol lacking 5-->3 exonuclease.Select against parental strands with Dpn1 restriction enzyme, which cuts methylated and hemimethylated DNA.TransformGeneral procedure

Method of mutagenesisA large amount of experimental procedures have been developed for directed mutageneis of cloned genes.All the procedures utilizes :A synthetic oligonucleotide complimentary to the area of the gene of interest but has the desired nucleotide change.What is an oligonucleotide?An oligonucleotide is a short piece of DNA usually 10-30 nt long.A vector e.g. a plasmid or M13.What is M13 ?

13Directed MutagenesisDirected mutagenesis can be done using:M13Plasmid DNAPCRRandom primersDegenerate primersNucleotide analogsError prone PCRDNA shuffling14

Directed Mutagenesis Using M13For the procedure the following must be known:The nucleotide sequence that encodes the mRNA codon to be changed. The amino acid changes that are to be made.The procedure involves:The gene of interest is inserted into the ds form of the M13 bacteriophage.(M13 has ssDNA and replicated via a dsDNA intermediate).The ssDNA is isolated from the M13 phage.15Directed Mutagenesis using M13The ssDNA is mixed with an excess of the synthetic oligonucleotide. The oligo is complimentary to the area of the cloned gene except for the one nucleotide to be changed.The oligo anneals to the ssDNA in the homologous region of the cloned gene.The oligo acts a primer for DNA synthesis using the M13 DNA as a template and the enzyme Klenow fragment of DNA polymerase I.T4 DNA ligase is used to ligate the 2 ends of the newly synthesized DNA.The newly synthesized M13 DNA is transformed into E. coli.16Directed Mutagenesis Using M13

17Directed Mutagenesis Using M13Because DNA replicates semi-conservatively half the cells should have the mutant gene.Mutant plaques are identified by DNA hybridization using the oligo as probe. Only 5% of the plaques carry the mutant gene. This makes isolation of those plaques with the mutant gene difficult.To produce large quantities of altered protein, the mutant gene is usually spliced from the M13 DNA by restriction enzymes and cloned into an E. coli plasmid.The procedure has been modified to to enrich for the number of mutant plaques.18Enrichment for the # of Mutant PlaquesOne strategy has been to introduce M13 vector carrying the desired gene into an E. coli strain with 2 defective enzymes:A defective form of dUTPase (dut). Cells with defective dUTPase has elevated levels of dUTP which is incorporated into the DNA often replacing dTTP.A defective Uracil N-glycosylase (ung). Uracil N-glycosylase is the enzyme that removes dUTP which is incorporated into DNA during replication.19Enrichment for the # of Mutant PlaquesThe procedure involves:The desired gene is cloned into M13 vector.The M13 vector with the desired gene is transformed into E. coli stain dut/ung, which produces ssDNA with 1% of the T replaced by U.An excess of oligonucleotide is added.The synthesis of a second strand occurs.20Enrichment for the # of Mutant PlaquesAddition of T4 ligase.The dsDNA is transformed into E. coli wild type strain.The wild type E. coli with functional ung gene will use Uracil N-glycosylase which will remove the dUTP which was incorporated into the DNA. Therefore the original DNA strand is degraded and only the mutant strand remains.In this way the number of plaques with the mutant gene is greatly increased.21Enrichment for the # of Mutant Plaques

22Oligonucleotide-Directed Mutagenesis Using Plasmid DNAOne of the disadvantages of performing directed mutagenesis using M13 vector is the large number of steps involved.That is: Clone the target gene into M13 vector.Transform into E.coli.Then reclone the gene into an E. coli plasmid.Why are all these steps necessary?23Oligonucleotide-Directed Mutagenesis Using Plasmid DNA.One approach includes: Inserting the desired gene into the multiple cloning site (mcs) of a plasmid vector. What is multiple cloning site (mcs) of a plasmid vector? Denaturation of the dsDNA of the plasmid by alkaline treatment i.e. dsDNA ssDNA. Why?Addition of 3 distinct oligonucleotide primers: One oligo is designed to alter the target gene.The second is designed to correct a mutation in an Amp resistant gene i.e amps ampr (SAR)The third oligo is designed to cause a mutation in a tet resistant gene i.e. tetr tets (RST)24Oligonucleotide-Directed Mutagenesis Using Plasmid DNAThe oligos are added along with 4 dNTPS and DNA polymerase.The oligos anneal and DNA polymerase synthesizes a new strand of DNA.T4 DNA ligase ligates the DNA.The rxn mixture is transformed into E. coli.Transformants are selected for ampr and tets. How?Using this method >90% of the transformants will have the mutation in the desired gene.The plasmid, E. coli, enzymes and 2 of the oligos are sold in a kit to facilitate wide spread use.25Oligonucleotide-Directed Mutagenesis Using Plasmid DNA

26Oligonucleotide-Directed Mutagenesis Using Plasmid DNA

If we did not have antibiotic markers how could we select for mutant gene?27PCR-amplified Oligonucleotide Directed MutagenesisPCR can be used to :Enrich for the mutant geneAvoid using M13 vectorThe procedure involves:The target gene is cloned into an E.coli plasmid.2 specific oligos are added to the PCR reaction.One primer is complimentary to the target.The other primer is complimentary to the target gene except for the nucleotide that is targeted for change.28PCR-amplified Oligonucleotide Directed MutagenesisThe oligos maybe overlapping.During PCR the complete target gene and plasmid are amplified.T4 ligase is added to the produce a circularized DNA from the linear PCR-amplified DNA.The recombinant plasmid is transformed into E. coli.Half the cells will have the mutant gene and half will have the wild type gene.The plasmid with the mutant gene can be identified by restriction digestion, PCR or DNA hybridization.29Directed-Mutagenesis using PCR

30Random Mutagenesis with Degenerate PrimersWhat is a random mutation?So far we have discussed directed mutagenesis at a pre-determined site in a cloned gene.Random mutagenesis involves mutation at any site in the DNA.Random mutagensis is useful because sometimes it is not known which specific nucleotide change that will produce the desired protein.What is a degenerate primer?A degenerate primer is an oligonucleotide where the nucleotides at some positions are varied.ATCCGATGGAATCisoleucineACCCGATAGAACCThreonineAGCCGATCGAAGCSerineAACCGATTGAAACAsparagine

31Random mutagenesis: Error Prone PCRSome heat stable DNA polymerases used during PCR can occasionally insert the wrong nucleotide generating mutations (Error Prone PCR).By modifying PCR conditions e.gDNA template concentrationAdding unequal concentration of each nucleotidesAdd Mn 2+ It is possible to introduce mutations into the PCR product.This product is then cloned and the modified protein expressed and tested for the desired properties.(3rd ed only)32Random Mutagenesis with Degenerate PrimersDegenerate primers can be used to introduce random mutations into a target gene.The procedure involves:Insertion of the target gene into a plasmid between two unique restriction sites.Using PCR in separate reactions to amplify overlapping fragments.This requires two pairs of primers (i.e. 4 primers) including 2 degenerate overlapping primers which anneal near the centre of the target gene.Two primers which anneals on opposite strands upstream the unique restriction sites. 33PCR-amplified Oligonucleotide Directed Mutagenesis

34Random Mutagenesis with Degenerate PrimersEach reaction has :1 degenerate primer (2, 4)1 primer upstream the restriction site (1, 3)After PCR the products are purified and combined.Denaturation and renaturation of the PCR products results in some DNA overlapping the target DNA.DNA polymerase is used to form complete dsDNA.This PCR product is digested with two restriction enzymes for which there are unique sites.The amplified DNA is cloned into a plasmid and transformed into E. coli which will express the modified protein.35PCR-amplified Oligonucleotide Directed Mutagenesis

36PCR-amplified Oligonucleotide Directed Mutagenesis

37Random Mutagenesis Using Nucleotide AnalogsWhat is a nucleotide?A unit of a nucleic acid consisting of a sugar, a base, and a phosphate.What is a nucleoside?A unit of a nucleic acid consisting of a sugar and a base.What is a nucleotide analog?A nucleotide analog is structurally similar to a nucleotide but is chemically different.E.g. 5 bromouracil is an analog of thymine.A nucleotide analog can be used to cause random mutations in DNA.38Nucleotide Analog

39Random Mutagenesis Using Nucleotide AnalogsThe procedure involves:The cloned gene is placed in a plasmid next to two closely placed restriction sites.The recombinant plasmid is treated with the two restriction enzymes to produce 5 and 3 recessed ends and 5 and 3 protruding ends.Recessed is the opposite of protruding, it simply means not sticking out or set back.The enzyme exonuclease III (Exo III) is added and will specifically degrade the DNA from the 3 recessed end only, but not from 5 recessed end or the protruding ends.40Random Mutagenesis Using Nucleotide Analogs

41Random Mutagenesis Using Nucleotide AnalogsAfter a specific time, the reaction is terminated and the gap produced is filled by Klenow fragment of DNA polymerase I.The dNTP mix used contains 4 normal nucleotides and one nucleotide analog.The nucleotide analog will be incorporated at several places along the DNA.T4 ligase is added to ligate the DNA.The recombinant plasmid with the nucleotide analog is transformed into E. coli.During replication in E. coli the nucleotide analog will direct the incorporation of bases distinct from that in the wild type gene creating random mutations through out the cloned gene.42Random Mutagenesis Using Nucleotide Analogs

43Improving StabilityProtein stability can be increased by creating a molecule which will not readily unfold under unfavorable conditions.Protein stability can be improved by:Adding disulphide bondsReplacing labile amino acidsReducing the number of free S-H (sulphydryl) groups. Adding disulphide bondsDisulphide bonds can significantly stabilize the native structure of proteins. This effect is presumed to be due to the decrease in configuration chain entropy of the unfolded polypeptide.Ex. Mutagenesis of LysozymeWild type lysozyme has 2 cysteine residues and no disulphide bonds.Site-directed mutagenesis was used to introduce new cysteine residues and new internal S-S bonds between amino acids:3 and 979 and 16421 and 142After mutagenesis each mutant gene was expressed in E. coli.The modified enzymes were purified and tested for enzyme activity and thermo stability.The results showed that the thermal stability increased with the presence of disulphide bonds.The most thermo stable mutant was the one with 3 S-S bonds.

46XylanaseSite-directed mutagenesis was used to produce 8 mutants xylanase proteins with increase S-S bonds and increase stability.3 of the mutants were as active as the wild type at 60C.One mutant with an S-S bond between the C and N terminal ends of the enzyme had twice the activity of the wild type at 60C.This mutant remained active for 2 hrs while the wild type lost all its activity after 30 min at 60C.47XylanaseXylanase is extensively used in bleaching industryThe stage of the process where the enzyme is added is immediately after hot alkaline treatment.Therefore a thermostable xylanase is required.One attempt to solve this problem was to produce a modified xylanase (Bacillus circulans) with increase thermal stability.48XylanaseSite-directed mutagenesis was used to produce 8 mutants xylanase proteins with increase S-S bonds and increase stability.3 of the mutants were as active as the wild type at 60C.One mutant with an S-S bond between the C and N terminal ends of the enzyme had twice the activity of the wild type at 60C.This mutant remained active for 2 hrs while the wild type lost all its activity after 30 min at 60C.49XylanaseSite-directed mutagenesis was used to produce 8 mutants xylanase proteins with increase S-S bonds and increase stability.3 of the mutants were as active as the wild type at 60C.One mutant with an S-S bond between the C and N terminal ends of the enzyme had twice the activity of the wild type at 60C.This mutant remained active for 2 hrs while the wild type lost all its activity after 30 min at 60C.Devlopment of antibiotic-overproducing strainsDevlopment of antibiotic-overproducing strains by site directed mutagenesis of rpsL gene in Streptomyces lividians ex:- streptomycin & puromomycinSite-Directed Mutagenesis of N5 Carboxyaminoimidazole Ribonucleotide Mutase(Class I PurE) in Bacillus anthracis. ex:- Cause mutation in the enzyme responsible for proliferation of B. anthrasis

Mutagenesis of Streptokinase

Plasmin cleaves Sk at Lys 59 and 386 and the 328 , has only 16% activity as the native molecule.

To make Sk less susceptible, Lys at 59 and 386 were changed to Glu by site directed mutagenesis.

Glu was chosen to replace Lys because the length of the side chain was similar and Glu does not have a +ve charge.

Furthermore the half life of Sk mutant increases and mutant was 21 fold more protease resistant than native protein.

Mutagenesis of Subtilisins

The x-ray crystallography structure of subtilisin and the amino acids involved in the Ca2+ binding was known.

Oligonucleotide mutagenesis was used to construct a mutant protein by deleting amino acids 75-83 that is responsible for Ca2+ binding.The mutants were assayed for enzyme activity and stability.

Alternative method for mutagenesisIn vitro mutagenesis using synthetic oligonucleotides.Synthesis of complete modified gene de novo.

Reference Yoshiko okamoto-Hosoya, Susumu Okamoto & Kozo Ochi * National food research Institute , Tsukuba, Ibaraki 305-8642, Japan. Megan Silas Department of Bioengineering, University of Illinois at Urbana ChampaignAbdelghani TTA, Kunamneni A, Ellaiah P (2005) Isolation and mutagenesis of streptokinase-producing bacteria. Am J Immunology 1(4):125129A. Volkov, F. Jordan, Evidence for intramolecular processing of subtilisin sequestered on a solid support, J. Mol. Biol. 262 (1996) 595599